The present invention concerns DNA sequences encoding for kynurenine-3-hydroxylase (kyn-3-OHase).
This enzyme is a flavin-containing monooxygenase which is localized in the outer mitochondrial membrane (Okamoto H., Yamamoto S., Nozaki M. and Hayaishi O. 1967. Biochem. Biophys. Res. Commun. 26: 309-314); it catalyses the 3 hydroxylation of L-kynurenine (L-kyn), an intermediate in the oxidative metabolism of tryptophan (DeCastro F. T., Price J. M. and Brown R. R., 1956. J. Am. Chem. Soc. 78: 2900-2904).
The kynurenine pathway (see the scheme below) is the major route of peripheral tryptophan metabolism in mammals: most of this metabolism takes place in the liver. The abbreviations used are the following: IDO, Indoleamine 2,3-dioxygenase; TDO, Tryptophan 2,3-dioxygenase; ED, 3-hydroxyanthranilate 3,4-dioxygenase; QPRT, Quinolinate phosphoribosyltransferase; ATP, Adenosine 5'-triphosphate; NAD, Nicotinamide adenine dinucleotide; NMN, Nicotinamide mononucleotide. ##STR1##
This pathway not only provides a route for total oxidation of tryptophan to acetyl-Co-A, but it is also responsible for the synthesis de novo of the nicotinamide nucleotide coenzymes NAD and NADP (Bender D. A. and McCreanor G. M. 1985. Biochem. Soc. Trans. 13:441-443).
Most of the current interest in this pathway arises from the observations that two intermediate metabolites, kynurenic acid (KYNA) and quinolinic acid (QUIN), seem to play a significant role in neurological diseases, the first acting as a neuroprotectant and the second as a neurotoxic agent. Kyn-3-OHase is the first enzyme in the route of production of QUIN.
The importance of QUIN as a neurotoxic agent was first evident from work by Lapin (1978. J. Neural. Trans. 42:37-43) who demonstrated that the administration of QUIN to rats caused convulsions. This was not sufficient to classify QUIN as a neurotoxin; its action in the central nervous system was better clarified when electrophysiological studies revealed that it was an agonist at the excitatory amino acid receptor sites normally activated by glutamate and aspartate (Stone T. W. and Perkins M. N., 1981. Eur J. Pharmacol. 72: 411-412).
Moreover, using the intrastriatal injection model, QUIN toxicity has been shown to be mediated through the N-methyl-D-aspartate (NMDA) receptors (Beal M., Kowall N., Swartz K. J., Ferrante R. J. and Martin J. B. 1989. J. Neurosci. 8: 3901-3908; Foster A. C., Gill R. and Woodruff G. N. 1988. J. Neurosci. 8: 4745-4754). Consistent with the involvment of NMDA receptors were the studies that showed reversal of QUIN-induced damage pathology by competitive NMDA antagonists (Foster A. C., Vezzani A., French E. D. and Schwarcz R. 1984. Neurosci. Lett. 48: 273-278; Leeson P. D., Baker R., Carling R. W., Curtis N. R., Moore K. W., Williams B. J. et al. 1991. J. Med. Chem. 34: 1243-1252).
It is becoming clear that some of the most important functions of the nervous system, such as synaptic plasticity and synapse formation, critically depend on the behavior of NMDA receptor channels and that neurological damages caused by a variety of pathological states can result from exaggerated activation of NMDA receptor channels (For a review see: Mori H. and Mishina M. 1995. Neuropharmacology 34: 1219-1237). Excessive activation of these receptors may play an important role in the neuronal injury associated with several disease states, including hypoxia-ischemia (Simon R. P., Swan J. H., Griffiths T. and Meldrum B. A. 1984. Science 226: 850-852), hypoglycemia (Wieloch T. 1985. Science 230: 681-683) and Huntington's disease (Schwarcz R., Whetsell W. O. Jr. and Mangano R. M. 1983. Science 219: 316-318. Koh J. Y., Peters S. and Choi D. W. 1986. Science 234: 73-76).
Assuming that QUIN is pathogenic for certain disorders, it is desirable to inhibit its formation. To accomplish this goal, knowledge must be gained about the enzymes that make QUIN and the sites at which the pathway is controlled.
In theory, QUIN could be formed in the brain in several ways (see the Kynurenine pathway above): from tryptophan, as in macrophages and in liver, or from kynurenine or 3-hydroxykynurenine which, having been formed peripherally, cross the blood-brain barrier to undergo final conversion to QUIN by brain kyn-3-OHase, kynureninase and 3-hydroxyanthranilate 3,4-dioxygenase (in contrast peripherally formed 3-hydroxyanthranilic acid enters the brain very poorly).
The optimal target for the design of inhibitors should be the rate limiting step through the pathway. Enzymatic studies in vitro on all the currently known enzymes along the two branches of the pathway have revealed that kyn-3-OHase, kynureninase and quinolinate phosphorybosiltransferase (see the kynurenine pathway) could contribute to determine the cerebral QUIN levels, though it is still unclear which of these enzymes is most effective in the normal brain and under pathological conditions. Studies on QUIN level, after administration to mice of different precursors (Erickson J. B., Flanagan E. M., Chang S. Y., Salter M. and Reinhard J. F. Jr. 1992. Soc. Neurosci. Abstr. 18: 442.) showed that brain and serum QUIN arise from qualitatively similar pathways. However, brain QUIN appears to be controlled heavily at the kyn-3-OHase step. As a consequence of these studies kyn-3-Ohase can be considered an important enzyme target for inhibition of QUIN biosynthesis.
Furthermore, being the first enzyme in the route of production of QUIN, the inhibition of kyn-3-OHase could lead to an accumulation of KYNA, the neuroprotectant metabolite of the pathway.
KYNA is an effective excitatory amino acid receptor antagonist with a particularly high affinity to the glycine modulatory site. (J. Neurochem., 52, 1319-1328, 1989). As a naturally occurring brain metabolite (J. Neurochem., 51, 177-180, 1988 and Brain Res., 454, 164-169, 1988), KYNA probably serves as a negative endogenous modulator of cerebral glutamatergic function (Ann. N.Y. Acad. Sci., 11, 290-296,1990); while applied directly into the brain, it exhibits anticonvulsant and neuroprotective properties (Neurosci. Lett. 48: 273-278. 1984).
In confirmation of all these data kyn-3-OHase inhibitors have recently been successfully applied to demonstrate for the first time, in the rat brain in vivo, the functional interdependence of the two branches of the kynurenine pathway by shifting cerebral metabolism towards an enhanced production of KYNA (Neuroscience 61: 237-244. 1994; Soc. Neurosci. Abstr. 21, 436.3. 1995).
In particular, systemic administration of the new and potent kyn-3-OHase inhibitor (R,S)-3,4-dichlorobenzoylalanine (FCE 28833) (see also Example 3b) causes a dose-dependent elevation in endogenous kynurenine and KYNA levels in rat brain tissue (Speciale et al. Eur. J. of Pharmacology, vol. 315, p.263-267, 1996).
These chemicals clearly hold great promise as research tools and may also harbor therapeutic potential since a decrease in brain QUIN and a concomitant increase in brain KYNA could be clinically desirable.
Moreover variants of the kyn-3-OHase enzyme could be present in different tissues and organs, and may constitute a possible target to develop more specific drugs.
In this perspective it is fundamental to clone the gene encoding for kyn-3-OHase, so that the protein can be studied from a molecular point of view and the recombinant enzyme can be obtained in reasonable quantity for further studies.
The purification of the enzyme and at least the partial sequence of the protein are the first steps to design degenerate oligonucleotides and to proceed with the cloning of the gene in a conventional hybridization way.
In 1975 Nisimoto et al. described the isolation of kyn-3-OHase from the mitochondrial outer membrane of rat liver in a 5 steps purification procedure (Nisimoto Y., Takeuchi F. and Shibata Y. 1975. J. Biochem. 78: 573-581). They described the isolation of a single homogeneus protein with a molecular weight of 200,000 Da and an isoelectric point of pH 5.4. The purified enzyme had a specific activity of 140 nmol min.sup.-1 mg.sup.-1 and an overall yield of 0.04%. In 1979, the same authors reported a 3 steps purification procedure with a better overall yield but a lower specific activity (Nisimoto et al. J. of Chromatography 169: 357-364. 1979).
More recently any attempts to reproduce these results, also from our biochemistry laboratories, have been unsuccessful; this is probably due to an instability of the enzyme during purification that do not allow a further analysis of its amino acidic sequence, thus precluding the possibility to adopt a conventional cloning approach.
Therefore, there is the need to set up an alternative cloning procedure and the invention aims to achieve this goal. According to the invention the problem is solved through the functional expression cloning in Xenopus laevis oocytes; this method is particularly useful to overcome the inconveniences deriving from the isolation of the protein by biochemical methods.
The functional expression cloning was first pioneered by Noma et al. (Noma Y., Sideras P., Naito T., Bergstedt-Lindquist S., Azuma C., Severinson E., Tanabe T., Kinashui T., Matsuda P., Yaoita Y. and Honjo T. 1986. Nature 319: 640-646.) and has been rapidly applied to the cloning of several plasma membrane proteins.
The strategy of functional expression cloning can be summarised as follow: functional expression, obtained with total mRNA from a given source, confirms the presence of mRNA coding for the protein of interest. The mRNA can then be fractionated according to size, the active fraction identified, and, after reverse transcription, used for the generation of a cDNA library. This library is then transcribed into polyadenylated cRNA, capped and expressed in the Xenopus oocytes. Subsequent appearance of the function in the oocytes confirms the presence of a full length clone (or at least a consistent functional part of it) in the library.
Functional expression cloning has the advantage over classical strategies in that the danger of sequencing "false positive" clones, due to cross reactivity of antibodies or nucleotide probes, can be avoided. Furthermore, most of the times full-length clones will directly be obtained (Sigel E. 1990. J. Membrane Biol. 117: 201-221).
Once cloned, the kyn-3-OHase cDNA obtained from a given source can be used to prepare probes for the screening of libraries derived from a different organisms. In this way, the expert in the art will recognize that, on the basis of the information provided herein, enzymes homologous to the ones specifically disclosed herein can be readily identified.